Polysialic acid is an important virulence factor for most of the capsular types of Escherichia coli and Neiseria meningitidis causing meningitis in infants and children. This poorly immunogenic polysaccharide plays a role in neural tissue development. The enzymes involved in the synthesis of polysialic acid in bacteria are being identified, purified and characterized. Common features between these enzymes and anologous enzymes in sialylation of mammalian glycoconjugates are being determined. Bovine pituitary CMP-sialic acid synthetase was partially purified at the time of the previous report. During this reporting period the CMP-sialic acid synthetase was purified further on a sialic acid based affinity column. The b-allyl ketoside of sialic was synthesized. This residue was coupled to activate agarose via a cysteamine linker. Enzyme eluting from this resin exhibited a major band of 60,000 on SDS. The enzyme activity eluted as 150,000 MW on gel filtration also eluted as 60,000 on SDS.A strain of CHO cells used to manufacture recombinant DNAase failed to properly sialylate glycoproteins. While this mutant strain does not produce CMP-sialic acid it does contain an active enzyme. Efforts are underway to express E. coli CMP-sialic acid synthetase in CHO mutants.Mutants in the E.coli K1 neuB gene cannot make sialic acid and thus require an addition of sugar for polysialic acid synthesis. The neuB gene was cloned into an expression vector and the enzyme purified to homogeniety. The amino terminal sequence of the purified enzyme was determed to be the same as that deduced from the nucleotide sequence of the neuB gene. The enzyme was shown to form sialic acid from N- acetylmannosamine and phosphoenol pyruvate. Efforts to further characterize this enzyme are underway and to use this enzyme to synthesize polysialic acid analogues.